Transcription initiation factor TFIIB recruits RNA polymerase II into the promoter region by interacting with the preformed TFIID-TATA box complex, thus specifying the transcription initiation site. Moreover, it has been proposed that TFIIB, as well as TFIID, is a target for a transcriptional activator. Therefore, exploring the mechanism by which TFIIB interacts with the TFIID-TATA box complex is especially important to understand transcription. Through mutational analysis it has been determined that only the C-terminal two-thirds of TFIIB, which contains the sigma-factor sequence similarities, two basic repeats and direct repeats, is sufficient for interaction with the TFIID-TATA box complex. However, an extra N-terminal 84 amino acid region, which does not contain any obvious known motifs, is required for transcription activity. Moreover, amino acid substitution analysis indicates that the second basic repeat of TFIIB is an important domain for interaction with the TFIID-TATA box complex. A reconstituted system containing a partially purified TFIID from Drosophila embryos stimulates transcription activity in vitro but a system containing a TATA box-binding subunit of TFIID (TFIIDtau) does not respond to trans-activators, although TFIIDtau binds to a TATA box and catalyzes basal level of transcription. Using anti-TFIIDtau immunoaffinity chromatography, 9 polypeptides (210k, 110k, 85k, 62k, 58k, 42k, 28k, 22k, 21kDa) were identified as native Drosophila TFIID components that are tightly associated with TFIIDtau. To analyze the functional activity of purified TFIID components, template DNA and other transcription factors were reconstituted with purified TFIID on an antibody-Sepharose resin. Immobilized TFIID mediates not only basal levels of transcription but upstream-stimulating factor (USF) activation as well. These results suggest that one or more of these additional polypeptides are required as functional TFIID subunits to regulate transcription by USF in cooperation with TFIIDtau.